The obligate intracellular parasite Toxoplasma gondii can infect an extremely broad range of host species. This promiscuity may be enabled by the parasite's ability to inject its own invasion proteins into target cells. To invade a host cell the infective form of the parasite, the tachyzoite, attaches to a target cell, injects parasite protein into that cell, and then enters the cell, creating the parasitophorous vacuole in which it will replicate. The proteins that are introduced into the target cell are stored in specialized vacuolar organelles called rhoptries. Some of the rhoptry proteins are required for formation of the moving junction, a structure that is essential for invasion and may propel the parasite into the host cell. Host cell defenses are modified by other injected rhoptry proteins, such as protein kinases that act on host STAT and p47 GTPase functions. Despite the importance of these rhoptry proteins, it is completely unknown how they are introduced into the host cell. I hypothesize that there is a specific protein structure that is required for injection of parasite proteins into host cells, and that it will be possible to generate temperature-sensitive mutants where that structure is disrupted. The goal of this project is to discover the mechanism of protein injection by looking for a specific class of invasion mutants. To find genes that are essential for invasion, I will isolate and identify temperature-sensitive mutants of Toxoplasma gondii that are defective in host cell invasion. I will then determine whether any of these mutants have a defect in protein injection into the host cell, using a previously published FRET-disruption assay. Characterization and sequencing of identified injection mutants will allow me to identify important new elements of the host cell invasion machinery. Once injection proteins are identified they will be tagged for localization and affinity purification to identify binding partnrs. This machinery is likely to be conserved throughout the Apicomplexa family, just as the invasion machinery is, and proteins identified here may help us understand the invasion of Plasmodium falciparum as well.